Diagnosis and treatment of refractory infectious diseases using nanopore sequencing technology:Three case reports

BACKGROUND Infectious diseases are still one of the greatest threats to human health,and the etiology of 20%of cases of clinical fever is unknown;therefore,rapid identification of pathogens is highly important.Traditional culture methods are only able to detect a limited number of pathogens and are time-consuming;serologic detection has window periods,false-positive and false-negative problems;and nucleic acid molecular detection methods can detect several known pathogens only once.Three-generation nanopore sequencing technology provides new options for identifying pathogens.CASE SUMMARY Case 1:The patient was admitted to the hospital with abdominal pain for three days and cessation of defecation for five days,accompanied by cough and sputum.Nanopore sequencing of the drainage fluid revealed the presence of orallike bacteria,leading to a clinical diagnosis of bronchopleural fistula.Cefoperazone sodium sulbactam treatment was effective.Case 2:The patient was admitted to the hospital with fever and headache,and CT revealed lung inflammation.Antibiotic treatment for Streptococcus pneumoniae,identified through nanopore sequencing of cerebrospinal fluid,was effective.Case 3:The patient was admitted to our hospital with intermittent fever and an enlarged neck mass that had persisted for more than six months.Despite antibacterial treatment,her symptoms worsened.The nanopore sequencing results indicate that voriconazole treatment is effective for Aspergillus brookii.The patient was diagnosed with mixed cell type classical Hodgkin's lymphoma with infection.CONCLUSION Three-generation nanopore sequencing technology allows for rapid and accurate detection of pathogens in human infectious diseases.

Advances in single-cell sequencing technology in microbiome research

With the rapid development of histological techniques and the widespread applica-tion of single-cell sequencing in eukaryotes,researchers desire to explore individual microbial.genotypes and functional expression,which deepens our understanding of microorganisms.In this review,the history of the development of microbial detection technologies was revealed and the difficulties in the application of single-cell sequencing in microorganisms were dissected as well.Moreover,the characteristics of the currently emerging microbial single-cell sequencing(Microbe-seq)technology were summarized,and the prospects of the application of Microbe-seq in microorganisms were distilled based on the current development status.Despite its mature development,the Microbe-seq technology was still in the optimization stage.A retrospective study was conducted,aiming to promote the widespread application of single-cell sequencing in microorganisms and facilitate further improvement in the technol-ogy.

Application of Biological Nanopore Sequencing Technology in the Detection of Microorganisms

Environmental pollution and the spread of pathogenic microorganisms pose a significant threat to the health of humans and the planet.Thus,understanding and detecting microorganisms is crucial for maintaining a healthy living environment.Nanopore sequencing is a single-molecule detection method developed in the 1990s that has revolutionized various research fields.It offers several advantages over traditional sequencing methods,including low cost,label-free,time-saving detection speed,long sequencing reading,real-time monitoring,convenient carrying,and other significant advantages.In this review,we summarize the technical principles and characteristics of nanopore sequencing and discuss its applications in amplicon sequencing,metagenome sequencing,and whole-genome sequencing of environmental microorganisms,as well as its in situ application under some special circumstances.We also analyze the advantages and challenges of nanopore sequencing in microbiology research.Overall,nanopore sequencing has the potential to greatly enhance the detection and understanding of microorganisms in environmental research,but further developments are needed to overcome the current challenges.

The next-generation sequencing technology and application

As one of the key technologies in biomedical research,DNA sequencing has not only improved its productivity with an exponential growth rate but also been applied to new areas of application over the past few years.This is largely due to the advent of newer generations of sequencing platforms,offering ever-faster and cheaper ways to analyze sequences.In our previous review,we looked into technical characteristics of the nextgeneration sequencers and provided prospective insights into their future development.In this article,we present a brief overview of the advantages and shortcomings of key commercially available platforms with a focus on their suitability for a broad range of applications.

The History and Advances of Reversible Terminators Used in New Generations of Sequencing Technology

DNA sequencing using reversible terminators, as one sequencing by synthesis strategy, has garnered a great deal of interest due to its popular application in the second-generation high-throughput DNA sequencing technology. In this review, we provided its history of develop- ment, classification, and working mechanism of this technology. We also outlined the screening strategies for DNA polymerases to accommodate the reversible terminators as substrates during polymerization; particularly, we introduced the ＂REAP＂ method developed by us. At the end of this review, we discussed current limitations of this approach and provided potential solutions to extend its application.

Application of high-throughput sequencing technology in HIV drug resistance detection

Drug resistance via drug-resistant mutations in the human immunodeficiency virus(HIV)genome is the primary cause of antiviral therapy failure.Consequently,HIV drug resistance genotyping has become a critical approach in HIV prevention and control.Compared to the Sanger sequencing technology,high-throughput sequencing(HTS)technology has superior sensitivity and timeliness,with strong detection capabilities for low-frequency mutations.With the continued advancement of HTS technologies,their prominence in HIV drug resistance detection techniques has increased accordingly.This article will review the latest developments in HTS technology and its applications in HIV drug resistance testing.

Corrigendum to Nanopore-based Fourth- generation DNA Sequencing Technology＇ [GPB 144 （2015）- GPB 13]1 （4-16）]

The authors regret that there was an incorrect citation in the Table 1 of the article ＂Nanopore-based Fourth-generation DNA Sequencing Technology＂, which was published in the journal Genomics, Proteomics ＆ Bioinformatics, Issue 1, 2015. In the table, the image in the 4th row originally appeared in the article ＂Nanopore-based sequence-specific detection of duplex DNA for geno- mic profiling＂ published by Nano Letters in 2010. Therefore, Ref. [8], which was associated with the image in the current Table l, should be replaced by the reference： Singer A, Wanunu M, Morrison W, Kuhn H, Frank-Kamenetskii M, Meller A. Nanopore- based sequence specific detection of duplex DNA for genomic profiling. Nano Lett 2010; 10：738-42.＂ The copy right permission of this article has been obtained. The authors would like to apologize for any inconvenience caused.

Corrigendum to ‘Nanopore-based Fourth-generation DNA Sequencing Technology' [GPB 144(2015)–GPB 13/1(4–16)]

The authors regret that there were typos in the online version of Figure 4 published in Issue 1,2015.In the figure legend boxes of panels F and G,‘‘Ni’’should be corrected to‘‘N’’for the labeling of the green curves.The figure in the printed version has been corrected.The correct Figure 4 is shown below.The authors would like to apologize for any inconvenience caused.

Streptococcus anginosus in the development and treatment of precancerous lesions of gastric cancer

The microbiota is strongly association with cancer.Studies have shown significant differences in the gastric microbiota between patients with gastric cancer(GC)patients and noncancer patients,suggesting that the microbiota may play a role in the development of GC.Although Helicobacter pylori(H.pylori)infection is widely recognized as a primary risk factor for GC,recent studies based on microbiota sequencing technology have revealed that non-H.pylori microbes also have a significant impact on GC.A recent study discovered that Streptococcus anginosus(S.anginosus)is more prevalent in the gastric mucosa of patients with GC than in that of those without GC.S.anginosus infection can spontaneously induce chronic gastritis,mural cell atrophy,mucoid chemotaxis,and heterotrophic hyperplasia,which promote the development of precancerous lesions of GC(PLGC).S.anginosus also disrupts the gastric barrier function,promotes the proliferation of GC cells,and inhibits apoptosis.However,S.anginosus is underrepresented in the literature.Recent reports suggest that it may cause precancerous lesions,indicating its emerging pathogenicity.Modern novel molecular diagnostic techniques,such as polymerase chain reaction,genetic testing,and Ultrasensitive Chromosomal Aneuploidy Detection,can be used to gastric precancerous lesions via microbial markers.Therefore,we present a concise summary of the relationship between S.anginosus and PLGC.Our aim was to further investigate new methods of preventing and treating PLGC by exploring the pathogenicity of S.anginosus on PLGC.

Overlapping infections of Mycobacterium canariasense and Nocardia farcinica in an immunocompetent patient:A case report

BACKGROUND Infections by non-tuberculous mycobacteria(NTM)have become more common in recent years.Mycobacterium canariasense(M.canariasense)was first reported as an opportunistic pathogen in 2004,but there have been very few case reports since then.Nocardia is a genus of aerobic and Gram-positive bacilli,and these species are also opportunistic pathogens and in the Mycobacteriales order.Conventional methods for diagnosis of NTM are inefficient.Metagenomic next-generation sequencing(mNGS)can rapidly detect many pathogenic microorganisms,even rare species.Most NTM and Nocardia infections occur in immunocompromised patients with atypical clinical symptoms.There are no previous reports of infection by M.canariasense and Nocardia farcinica(N.farcinica),especially in immunocompetent patients.This case report describes an immunocompetent 52-year-old woman who had overlapping infections of M.canariasense,N.farcinica,and Candida parapsilosis(C.parapsilosis)based on mNGS.CASE SUMMARY A 52-year-old woman presented with a productive cough and chest pain for 2 wk,and recurrent episodes of moderate-grade fever for 1 wk.She received antibiotics for 1 wk at a local hospital,and experienced defervescence,but the productive cough and chest pain persisted.We collected samples of a lung lesion and alveolar lavage fluid for mNGS.The lung tissue was positive for M.canariasense,N.farcinica,and C.parapsilosis,and the alveolar lavage fluid was positive for M.canariasense.The diagnosis was pneumonia,and application of appropriate antibiotic therapy cured the patient.CONCLUSION Etiological diagnosis is critical for patients with infectious diseases.mNGS can identify rare and novel pathogens,and does not require a priori knowledge.

Current status and future perspectives for sequencing livestock genomes

Only in recent years, the draft sequences for several agricultural animals have been assembled. Assembling an individual animal＇s entire genome sequence or specific region（s） of interest is increasingly important for agricultura researchers to perform genetic comparisons between animals with different performance. We review the current status for several sequenced agricultural species and suggest that next generation sequencing （NGS） technology with decreased sequencing cost and increased speed of sequencing can benefit agricultural researchers. By taking advantage of advanced NGS technologies, genes and chromosomal regions that are more labile to the influence of environmental factors could be pinpointed. A more long term goal would be addressing the question of how animals respond at the molecular and cellular levels to different environmental models （e.g. nutrition）. Upon revealing important genes and gene-environment interactions, the rate of genetic improvement can also be accelerated. It is clear that NGS technologies will be able to assist animal scientists to efficiently raise animals and to better prevent infectious diseases so that overall costs of animal production can be decreased.

A KIND OF PAPR REDUCTION METHOD BASED ON PRUNING WPM AND PTS TECHNOLOGY

Wavelet packet multicarrier system gains widespread concern because of its better resistance performance to Inter-Symbol Interference (ISI) and Inter-Carrier Interference (ICI), as well as the higher spectrum efficiency. However, multicarrier system has a high Peak to Average Power Ratio (PAPR), which will lead to many problems such as lower system performance. In order to solve the problem, a kind of PAPR reduction method based on pruning Wavelet Packet Modulation (WPM) and Partial Transmit Sequences (PTS) technology is proposed in this paper, through proper pruning of the full-tree structure of wavelet packet modulation in the PTS technology to reduce the number of nodes in the system, and finally improve the reduction effect of PAPR. Simulation results show that when Complementary Cumulative Distribution Function (CCDF) is 10 -3 , PTS based on pruning WPM compared with PTS technique and pruning technique has improved about 1 dB and 1.5 dB, which will not affect the system's Bit Error Rate (BER) performance in the wavelet packet multicarrier system.

单细胞转录组测序技术畜牧业标准化现状分析

Single-cell RNA sequencing technology(scRNA-seq)is an emerging,high-throughput,high-resolution big data production and analysis technology,which has helped some pioneering research and discoveries in the field of animal husbandry in recent years.This technology provides an essential basis for the early selection of livestock and poultry and the selection of excellent characters,showing that it has great application potential and broad prospects in animal husbandry research and production.Although scRNA-seq technology has established a wide range of commercial production services at home and abroad,the standardization status of this technology in animal husbandry needs further exploration,as it can provide clues and foundations for the application of scRNA-seq technology in animal husbandry.

Diagnosis and treatment of Whipple disease after kidney transplantation:A case report

BACKGROUND Kidney transplantation is the standard treatment for end-stage renal disease.Particularly,rare and specific pathogenic infections which are asymptomatic are often difficult to diagnose,causing delayed and ineffective treatment and thus seriously affecting prognosis.Tropheryma whipplei(T.whipplei)is a Gram-positive actinomycete widely found in soil,sewage,and other external environments and is present in the population as an asymptomatic pathogen.There is relatively little documented research on T.whipplei in renal transplant patients,and there are no uniform criteria for treating this group of post-transplant patients.This article describes the treatment of a 42-year-old individual with post-transplant T.whipplei infection following kidney transplantation.CASE SUMMARY To analyze clinical features of Whipple’s disease and summarize its diagnosis and treatment effects after renal transplantation.Clinical data of a Whipple’s disease patient treated in the affiliated hospital of Guizhou Medical University were collected and assessed retrospectively.The treatment outcomes and clinical experience were then summarized via literature review.The patient was admitted to the hospital due to recurrent diarrhea for 1 mo,shortness of breath,and 1 wk of fever,after 3 years of renal transplantation.The symptoms of the digestive and respiratory systems were not significantly improved after adjusting immunosuppressive regimen and anti-diarrheal,empirical antibiotic treatments.Bronchoscopic alveolar fluid was collected for meta-genomic next-generation sequencing(mNGS).The deoxyribonucleic acid sequence of Tropheryma whipplei was detected,and Whipple’s disease was diagnosed.Meropenem,ceftriaxone,and other symptomatic treatments were given,and water-electrolyte balance was maintained.Symptoms resolved quickly,and the patient was discharged after 20 d of hospitalization.The compound sulfamethoxazole tablet was continued for 3 mo after discharge.No diarrhea,fever,and other symptoms occurred during the 6-month follow-up.CONCLUSION Whipple’s disease is rare,with no specific symptoms,which makes diagnosis difficult.Polymerase chain reaction or mNGS should be immediately performed when the disease is suspected to confirm the diagnosis.

Variations in microbial community during nitrogen removal by in situ oxygen-enhanced indigenous nitrogen-removal bacteria

In this study, the enclosure system exhibited perfect nitrogen removal performance with in situ oxygen-enhanced indigenous aerobic denitrifying bacteria in an enclosure experiment. We explored changes in the microbial community during the nitrogen removal process using the MiSeq high-throughput sequencing technology. The results revealed a total of 7974 and 33653 operational taxonomic units(OTUs) for water and sediment systems, respectively, with 97% similarity. The OTUs were found to be affiliated with eight main phyla(Proteobacteria, Actinobacteria, Cyanobacteria, Bacteroidetes, Planctomycetes, Chloroflexi, Firmicutes, and Actinobacteria). The diversity of the enhanced system was found to be higher than that of the control system. Principal component analysis(PCA) revealed that significant spatial and temporal differences were exhibited in the microbial community during nitrogen removal in the enclosure experiment. Redundancy analysis(RDA)indicated that physical parameters(temperature, dissolved oxygen, and pH), nitrogen(total nitrogen and nitrate), functional genes(nirK and nirS), and dissolved organic carbon(DOC) were the most important factors affecting bacterial community function and composition. Lastly, the results suggested that the variation in the microbial community could be analyzed through the MiSeq high-throughput sequencing technology,which may provide technical support for future field tests.

Effect of a compound starter cultures inoculation on bacterial profile and biogenic amine accumulation in Chinese Sichuan sausages

Effects of a starter culture composed of Lactobacillus sakei,Pediococcus pentosaceus,Staphylococcus xylosus and S.carnosus at the ratios(m/m)of 1:1:1:1 on bacterial profile and biogenic amine accumulation in Chinese Sichuan sausages were demonstrated.In starter culture-inoculated sausages,Lactobacillus spp.,Pediococcus spp.and Staphylococcus spp.were predominant bacterial genus and their relative abundances ranged from 70.14%to 25.98%,57.66%to 14.08%,and 15.71%to 13.40%during fermentation,respectively.Accordingly,Lactobacillus spp.and Weissella spp.were predominant bacterial genus and their relative abundances ranged from 63.14%to 24.70%and 49.40%to 11.96%during spontaneous fermentation,respectively.Furthermore,the abundances of undesirable microorganisms such as Yersinia spp.,Enterobacter spp.,Acietobacter spp.and Psychrobacter spp.were lower than that of the control.The levels of histamine,putrescine,tyramine and cadaverine in Chinese Sichuan sausages with starter cultures inoculation were significantly lower(P<0.05)than that of the control,and were decreased by 83.09%,69.38%,51.87%and 57.20%,respectively,at the end of the ripening.These results revealed that the starter cultures inoculation was a better alternative for microbial quality improvement and biogenic amine reduction of Chinese Sichuan sausages with good sensory attributes.

Prediction of candidate small non-coding RNAs inAgrobacterium by computational analysis

Small non-coding RNAs with important regulatory roles are not confined to eukaryotes. Recent work has uncovered a growing number of bacterial small RNAs （sRNAs）, some of which have been shown to regulate critical cellular processes. Computational approaches, in combination with molecular experiments, have played an important role in the identification of these sRNAs. At present, there is no information on the presence of small non-coding RNAs and their genes in the Agrobacterium tumefaciens genome. To identify potential sRNAs in this important bacterium, deep sequencing of the short RNA populations isolated from Agrobacterium tumefaciens C58 was carried out. From a data set of more than 10,000 short sequences, 16 candidate sRNAs have been tentatively identified based on computational analysis. All of these candidates can form stem-loop structures by RNA folding predictions and the majority of the secondary structures are rich in GC base-pairs：：Some are followed by a short stretch of U residues, indicative of a rho-independent transcription terminator, whereas some of the short RNAs are found in the stem region of the hairpin, indicative of eukaryotic-like sRNAs. Experimental strategies will need to be used to verify these candidates. The study of an expanded list of candidate sRNAs in Agrobacterium will allow a more complete understanding of the range of roles played by regulatory RNAs in prokaryotes.

Polymer translocation through nanopore under external electric field:dissipative particle dynamics study

The DNA sequencing technology has achieved a leapfrog development in recent years. As a new generation of the DNA sequencing technology, nanopore sequenc- ing has shown a broad application prospect and attracted vast research interests since it was proposed. In the present study, the dynamics of the electric-driven translocation of a homopolymer through a nanopore is investigated by the dissipative particle dynam- ics （DPD）, in which the homopolymer is modeled as a worm-like chain （WLC）. The DPD simulations show that the polymer chain undergoes conformation changes during the translocation process. The different structures of the polymer in the translocation process, i.e., single-file, double folded, and partially folded, and the induced current block- ades are analyzed. It is found that the current blockades have different magnitudes due to the polymer molecules traversing the pore with different folding conformations. The nanoscale vortices caused by the concentration polarization layers （CPLs） in the vicinity of the sheet are also studied. The results indicate that the translocation of the polymer has the effect of eliminating the vortices in the polyelectrolyte solution. These findings are expected to provide the theoretical guide for improving the nanopore sequencing tech- nique.

Single-cell sequencing analysis of the maternal-fetal interface

The microenvironment at the maternal-fetal interface is optimized to facilitate the development and survival of the fetus during pregnancy.It involves a balance between cell populations and interactions of the fetal placenta with various cell types(ie,stromal cells,endothelial cells,immune cells,and fibroblasts)that are embedded in the maternal endometrium/decidua.Aberrant shifts in cell populations and deranged cell-cell interactions are closely related to pregnancy disorders.Thus,analysis of the dynamic changes in cell populations and their interactions at the maternal-fetal interface in normal and complicated pregnancies is essential to provide insights into the fundamental processes involved in the establishment and maintenance of normal pregnancy,and how these processes are dysregulated.Thus,informing novel pathways for therapeutic targets of pregnancy complications.Single-cell sequencing(SCS)is a powerful tool for transcriptome analysis at single-cell resolution.Combined with information on the developmental trajectory and function of different cell populations,SCS can provide an unparalleled opportunity for refining the spatiotemporal cell atlas to elaborate dynamic changes in cell populations and their interactions in tissues that consist of highly heterogeneous cell populations such as the maternal-fetal interface.This minireview briefly summarizes traditional methods and their limitations for analyzing maternal-fetal interface cell-cell interactions,and introduces the current applications,advantages,limitations,and prospective applications of SCS in research on maternal-fetal interactions.

Gut microbiome in allogeneic hematopoietic stem cell transplantation and specific changes associated with acute graft vs host disease

Allogeneic hematopoietic stem cell transplantation(aHSCT)is a standard validated therapy for patients suffering from malignant and nonmalignant hematological diseases.However,aHSCT procedures are limited by potentially life-threatening complications,and one of the most serious complications is acute graft-versus-host disease(GVHD).During the last decades,DNA sequencing technologies were used to investigate relationship between composition or function of the gut microbiome and disease states.Even if it remains unclear whether these microbiome alterations are causative or secondary to the presence of the disease,they may be useful for diagnosis,prevention and therapy in aHSCT recipients.Here,we summarized the most recent findings of the association between human gut microbiome changes and acute GVHD in patients receiving aHSCT.